Pharmacological agents are being developed to modulate phosphotyrosyl (pTyr) dependent cell signalling. Emphasis is on inhibitors of pTyr-dependent binding interactions which are mediated by src homology 2 (SH2) domains and on protein- tyrosine phosphatase (PTP) inhibitors. Central to both of these efforts is the development of new pTyr mimetics which afford either increased stability toward enzymatic degradation by PTPs or increased affinity. In the SH2 domain area, development of cell-permeable growth factor receptor-bound 2 (Grb2) antagonists is being undertaken as potential new therapeutics for a variety of cancers including erbB-2 and Met dependent cancers. A series of new pTyr-mimicking amino acid analogues, including non phosphate-containing analogues, have been prepared and incorporated into a tripeptide platform. Among these are, non phosphate containing analogues which exhibit low nanomolar Grb2 SH2 domain inhibition constants. Promising analogues exhibit potent inhibition of Grb2 binding in whole cell systems, and display good cytostatic effects against breast cancer cells grown in culture or in soft agar. Studies are currently underway to examine the utility of these agents in combination therapies directed against breast cancer. Preliminary cell studies indicate that non toxic concentrations of these synthetic Grb2 inhibitors can act cooperatively with certain standard cytotoxic chemotherapeutic agents, to significantly reduce the growth inhibitory dose. In cellular studies, these synthetic Grb2 inhibitors inhibit human growth factor (HGF)-induced cell migration in Met-containing fibroblasts at nanomolar concentrations and inhibit tubule formation potentially involved in angiogenesis. Work is currently in progress to examine these agents in whole animal metastasis models. Synthetic structural studies have produced novel conformationally constrained variants of lead Grb2 SH2 domain inhibitors. Initial data indicate that macrocyclization of tripeptide inhibitors can result in significant potency enhancement. Macrocycles from this series have exhibited the highest potency of any compounds yet tested in our erbB-2 dependent breast cancer systems. Work continues on the design, synthesis and biological evaluation of new macrocyclic Grb2 SH2 domain inhibitors. In the phosphatase area, a structure-based approach toward inhibitor design is being pursued. Using an epidermal growth factor receptor (EGFr)-derived pTyr-containing peptide sequence as a platform, we have examined a large number of novel non phosphorus containing pTyr mimetics for inhibitory potency against PTP1B. Highly potent motifs identified in this fashion have served as models for small molecule peptidomimetic design. In collaboration with scientists at Oxford University, we have obtained X-ray crystal structures of several of our peptidomimetic inhibitors bound to the PTP1B enzyme. Utilizing key binding elements evident in one of these crystal structures, and we have used focussed library methodology to enhance potency approximately 100-fold relative to the lead inhibitor. In separate work, information derived from the X-ray crystal structure of a lead inhibitor bound to PTP1B has been used to derive 2,6-naphthalene dicarboxylic acid as a new PTP-binding motif. The aim of this work is to identify high affinity small molecule inhibitors with improved bioavailability as tools for studying cellular signal transduction, and as potential therapeutic agents.